The human immunodeficiency viruses infect CD4.sup.+ macrophages and T helper cells. Although HIV-1 entry requires cell surface expression of CD4, to which the viral envelope glycoproteins bind, several studies have suggested that it is not sufficient for fusion of the viral envelope to the cellular plasma membrane. Early studies have shown that while human cells expressing a transfected CD4 gene were permissive for virus entry, murine cells expressing human CD4 were not. These findings led to the suggestion that there is a species-specific cell surface cofactor required in addition to CD4 for HIV-1 entry. Subsequent studies have shown that strains of HIV-1 that had been adapted for growth in transformed T-cell lines (T-tropic strains) could not infect primary monocytes or macrophages; in contrast, primary viral strains were found to infect monocytes and macrophages, but not transformed T cell lines. This difference in tropism was found to be a consequence of specific sequence differences in the gp120 subunit of the envelope glycoprotein, suggesting that multiple cell type-specific cofactors may be required for entry in addition to CD4.
The vast majority of people are susceptible to infection with HIV-1. However, rare individuals have been described that appear to remain uninfected by HIV-1 despite histories of multiple high-risk sexual exposures to the virus [Clerici et al., J. Infect. Dis. 165: 1012-1019 (1992); Langlade-Demoyen et al., J. Clin. Invest. 93: 1293-1297 (1994); Paxton et al., 1996, supra; Rowland-Jones et al., Nat. Med. 1: 59-64 (1995)]. In some cases this may simply be stochastic, or may be due to an extremely quiescent infection.
HIV-1 can broadly be divided into macrophage- or T-tropic isolates [Fisher et al., Nature 334: 444-447 (1988); Gartner et al., Science 233: 215-219 (1986)]; Koyanagi et al., Science 236: 819-822 (1987)]. Macrophage-tropic nonsyncytium-inducing (NSI) isolates infect primary macrophages but fail to infect transformed T-cell lines, while T-tropic syncytium-inducing (SI) strains have the reciprocal tropism. Both classes of HIV-1 efficiently infect CD4.sup.+ T-cells isolated from peripheral blood mononuclear cells (PBMC). Macrophage-tropic NSI viruses appear to be preferentially transmitted by sexual contact and constitute the vast majority of virus present in newly infected individuals [Zhu et al., Science 261: 1179-1181 (1993)]. The T-tropic SI viruses generally appear late in the course of infection during the so called "phenotypic switch" that often precedes the onset of AIDS symptoms [Connor and Ho, J. Virol., 68: 440-4408 (1994); Schuitemaker et al., J. Virol. 66, 1354-60 (1992)].
HIV-1 replication is initiated by attachment of the virus to the cell surface via high affinity binding of the envelope glycoprotein (Env) to CD4 on the cell surface [reviewed by [Sattentau and Weiss, Cell 52: 631-633 (1988)]. Subsequently, the viral envelope fuses to the cell membrane, depositing the viral core in the cytoplasm. The nature of the cofactors required for HIV entry proved elusive until the identification of specific receptor protein. The fusion reaction is mediated by newly-described seven transmembrane domain G protein-coupled molecules termed coreceptors [Alkhatib et al., Science 272: 1955-1958 (1996); Choe et al., Cell 85: 1135-1148 (1996); Deng et al., Nature, 381: 661-666 (1996); Doranz et al., Cell, 85: 1149-1158 (1996); Dragic et al., Nature 381: 667-673 (1996); Feng et al., Science 272: 872-877 (1996)]. The molecular basis of HIV-1 tropism appears to lie in the ability of Envs from macrophage-tropic and T-tropic viruses to interact with different coreceptors. T-tropic viruses tend to use fusin, a previously identified seven transmembrane protein related to the IL-8 receptor [Feng et al., 1996, supra]. Macrophage-tropic viruses primarily use CKR-5 (for C--C chemokine receptor-5), a seven transmembrane domain chemokine receptor [U.S. patent application Ser. No. 08/650,412 and Provisional Ser. No. 60/017,157, both filed May 20, 1996; and Ser. No. 08/666,020, and Provisional Ser. No. 60/020,043; Alkhatib et al., 1996, supra; Choe et al., 1996, supra; Deng et al., 1996, supra; Doranz et al., 1996, supra; Dragic et al., 1996, supra; Feng et al., 1996, supra]. Use of other chemokine receptors such as CKR-2B and CKR-3 by a minority of viruses has also been reported (Choe et al., 1996, supra; Doranz et al., 1996, supra).
Physiologically, chemokine receptors mediate the chemotaxis of T-cells and phagocytic cells to areas of inflammation [reviewed by Horuk, Trends Pharmacol. Sci. 15: 159-65 (1994)]. Upon ligand binding, the receptors transduce an intracellular signal that results in the rapid mobilization of intracellular calcium. Each of the eight known chemokine receptors is a G protein-coupled seven transmembrane domain protein with a characteristic pattern of ligand binding [reviewed by Schall, Cytokine 3: 165-183 (1991)]. CKR-5, which also serves as a major coreceptor for macrophage-tropic HIV-1, binds the .beta.-chemokines RANTES (regulated on activation, normal T expressed and secreted), MIP-1.alpha. (macrophage inflammatory protein) and MIP-10.beta. [Samson et al., 1996, supra]. The ligand for fusin has not yet been identified. High levels of RANTES, MIP-1.alpha. or MIP-1.beta. prevent replication of macrophage-tropic, but not T-tropic strains of HIV-1 [Cocchi et al., Science 720: 1811-1815 (1996)]. This inhibition is due to the binding of chemokines to CKR-5, resulting in a block to viral entry and fusion [Deng et al., 1996, supra; Dragic et al., 1996, supra]. The precise mechanism of this interference is unknown.
The nature of the cofactors required for HIV entry proved elusive until the recent identification by Feng et al. [Science 272, 872-877 (1996)] of fusin, a member of the seven transmembrane G-protein coupled receptor family. Fusin was shown to act as a co-receptor for T-tropic strains; however, it did not support infection of CD4.sup.+ cells by macrophage-tropic viruses, which more closely resemble those that predominate in infected individuals throughout the course of the disease, particularly in the asymptomatic phase. In addition, these strains appear to be responsible for HIV-1 transmission, both sexually and by transfer of infected blood. Rare individuals who are resistant to sexual transmission of HIV-1 have T-cells that are readily infected by T-tropic virus, but cannot be infected by macrophage-tropic virus, further supporting a role for macrophage-tropic virus in sexual transmission of HIV-1.
Cocchi et al. recently characterized inhibitors of HIV-1 replication present in supernatants of CD8.sup.+ T cells as the .beta.-chemokines RANTES, MIP-1.alpha. and MIP-1.beta. [Cocchi et al., (1996), supra]. Chemokines are chemotactic cytokines that are released by a wide variety of cells to attract macrophages, T cells, eosinophils, basophils and neutrophils to sites of inflammation (reviewed in ref. 14). The chemokines fall into two classes, C-X-C (.alpha.) and C--C (.beta.), depending on whether the first two cysteines are separated by a single amino acid or are adjacent. The .alpha.-chemokines such as IL-8, NAP-2 and MGSA are chemotactic primarily for neutrophils, while .beta.-chemokines such as RANTES, MIP-1.alpha., MIP-1.beta., MCP-1, MCP-2, and MCP-3 are chemotactic for macrophages, T-cells, eosinophils and basophils. The chemokines bind specific cell surface receptors belonging to the family of G protein-coupled seven transmembrane domain proteins (reviewed in Ref. 15). Upon binding their cognate ligands, chemokine receptors transduce an intracellular signal through the associated trimeric G protein. This results in a rapid increase in intracellular calcium concentration. There are at least seven human chemokine receptors that bind or respond to .beta.-chemokines with the following characteristic pattern: CC-CKR-1 (MIP-1.alpha., MIP-1.beta., MCP-3, RANTES), CC-CKR-2A and CC-CKR-2B (MCP-1, MCP-3), CC-CKR-3 (eotaxin, RANTES, MCP-3), CC-CKR4 (MIP-1.alpha., RANTES, MCP-1), CC-CKR-5 (MIP-.alpha., RANTES, MIP-1.beta.), and the Duffy blood group antigen (RANTES, MCP-1).
The cellular factors that account for the inability of macrophage-tropic HIV-1 to enter EU cells have not been elucidated. Importantly, the CD4.sup.+ T-cells of EU2 and EU3, while resistant to infection by macrophage-tropic virus, are readily infected by T-tropic HIV-1 [Paxton et al., 1996, supra]. Thus, the EU cells do not have a generalized inability to support virus replication. Presumably, they either lack a specific factor that is required for entry of macrophage-tropic HIV-1 or contain an inhibitor of this step of virus replication. T-cell clones derived from the PBMC of one of these individuals (EU2) generally secreted about 10-fold more .beta.-chemokine than similar clones derived from control individuals [Dragic et al., 1996, supra]. Thus, the resistance of these cells to HIV-1 infection could be caused by autocrine or paracrine blocking of CKR-5 coreceptor activity by the high levels of endogenous chemokines. Alternatively, genetic alteration of CKR-5 itself could decrease its ability to mediate viral entry.
Thus, there is a need in the art to understand the molecular mechanisms for resistance to macrophage-tropic HIV infection.
More particularly, there is a need in the art to identify the molecular basis for resistance to CKR-5 coreceptor-mediated HIV infection.
There is a further need in the art to distinguish autocrine or paracrine blocking of CKR-5 coreceptor activity from defective CKR-5.
These and other needs in the art are addressed by the present invention, as described below.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the application.